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Sennoside A, derived from the traditional Chinese medicine plant Rheum L., is a new dual HIV-1 inhibitor effective on HIV-1 replication.

ABSTRACT

Background: Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with new mode of action are still needed. An innovative approach is aimed to identify dual HIV-1 inhibitors, small molecules that can inhibit two viral functions at the same time. Rhubarb, originated from Rheum palmatum L. and Rheum officinale Baill., is one of the earliest and most commonly used medicinal plants in Traditional Chinese Medicine (TCM) practice. We wanted to explore TCM for the identification of new chemical scaffolds with dual action abilities against HIV-1.

Methods: R. palmatum L. and R. officinale Baill. extracts along with their main single isolated constituents anthraquinone derivatives were tested on both HIV-1 Reverse Transcriptase (RT)-associated DNA Polymerase (RDDP) and Ribonuclease H (RNase H) activities in biochemical assays. Active compounds were then assayed for their effects on HIV-1 mutated RTs, integrase (IN) and viral replication.

Results: Both R. palmatum L and R. officinale Baill. extracts inhibited the HIV-1 RT-associated RNase H activity. Among the isolated constituents, Sennoside A and B were effective on both RDDP and RNase H RT-associated functions in biochemical assays. Sennoside A was less potent when tested on K103N, Y181C, Y188L, N474A and Q475A mutated RTs, suggesting the involvement of two RT binding sites for its antiviral activity. Sennoside A affected also HIV-1 IN activity in vitro and HiV-1 replication in cell-based assays. Viral DNA production and time of addition studies showed that Sennoside A targets the HIV-1 reverse transcription process.

Conclusion: Sennoside A is a new scaffold for the development of HIV-1 dual RT inhibitors.

Keywords:

Sennoside A

HIV-1

Reverse transcriptase

Antiviral activity

Plant extracts

Dual inhibitor

Introduction

The Human Immunodeficiency Virus type 1 (HIV-1) encoded Reverse Transcriptase (RT) is responsible for the reverse transcription process of the viral genome, a fundamental step in the HIV-1 replication cycle and a still attractive target for the identification of new antiretroviral inhibitors (Esposito et al., 2012a; Corona et al., 2013; Distinto et al., 2013; Esposito and Tramontano, 2013). The HIV-1 RT is a multifunctional protein endowed of two main enzymatic functions: an RNA-Dependent DNA Polymerase (RDDP) activity, which accounts for the formation of the RNA: DNA intermediate, and a Ribonuclease H (RNase H) activity, involved in the hydrolytic cleavage of the RNA strand of the RNA: DNA hybrid (Esposito et al., 2012b). Currently, for the > 30 million of people worldwide infected by HIV-1 no vaccine is available, but an effective antiretroviral therapy has been developed, termed Highly Active Anti-Retroviral Therapy (HAART). HAART first line treatment is usually composed by a combination of two Nucleoside RT Inhibitors (NRTIs) to which a Non-Nucleoside RT Inhibitor (NNRTI) or a Protease Inhibitor (PI) is added. HIV-1 IN inhibitors (INIs) are expected to retain efficacy against HIV-1 strains resistant to other antiretroviral drugs, and represent a valid second-line therapy. The acute and chronic drug toxicities and the selection of drug resistant strains, in particular to RTIs, represent a valid reason to identify new antiviral agents. Indeed, the development of drugs with new mode of action, such as single molecules that may act on two different target enzymes or two different catalytic functions, would reduce the number of administered drugs, their chronic toxicity and the chance of selecting drug resistant viruses (Distinto et al., 2013; Esposito and Tramontano, 2013).

In the last 20 years, several research efforts have been focused on exploiting natural products as scaffolds for the development of potential antiviral agents (Li and Vederas, 2009; Cos et al., 2008; Yu et al., 2007; Bicchi et ai., 2009; Xu et al., 2015), although no natural compound able to block HIV-1 replication has ever entered clinical trials. In China, Traditional Chinese Medicine (TCM) is the major ancient therapeutic system and its herbal component is the most important one. Radix et Rhizoma Rhei (Da Huang in Chinese), usually originated from Rheum palmatum L. and R. officinale Baill., is one of the earliest and also the most commonly used medicinal plants in TCM practice (Beijing: Chinese Pharmaceutical Science and Technology Publishing House, 2010.

The earliest record of Rhubarb use appears to come from the Shen Nong Materia Medica (A. D. 102-200) of E. Han Dynasty and it was reported to be adopted to treat constipation and dysentery. Based on the modern investigations, the new application of Rhubarb involves cure of chronic renal failure, protection of damaged liver, and anti-aging effects (Xu et al., 2015; Lu et al., 2014; Xie and Sang, 2014). Rheum (Polygonaceae) includes more than 60 species distributed worldwide, with 39 species in the Western and Northern part of China, is one of the most important medicinal resources in China and one of the main plant sources containing natural anthraquinone compounds (Xiao et al., 1980). Among the anthraquinones reported to be present in Rhubarb (Ye et al., 2007), Sennosides (Sennoside A and Sennoside B) are considered to be the purgative components (Xiao et al., 1980; Zwaving, 1972), Rhein and Physcion possess antitumor and anti-inflammatory properties (Zwaving, 1972; He et al., 2011), with the latter presenting also hepatoprotective, antifungal and anticancer activities (He et al., 2011). Aloe-Emodin, Emodin and Chrysophanol have been reported to decrease the levels of serum total bilirubin, showing partial protective effects on cholestatic liver injury (Zhao et al., 2009). Glucosyl gallates (Maldonado et al., 2011), Naphthalenes (Tsuboi et al., 1977), and Catechins (Sill et al.. 1974) exhibited potential antioxidant and anticancer properties. A number of polyphenol derivatives, including, for instance, anthraquinones, have been reported to interfere with the life cycle of different viruses, among which encephalomyocarditis virus in mice (Barnard et al., 1992), human cytomegalovirus (Barnard et al., 1992; Barnard et al., 1995), poliovirus (Semple et al., 2011) and hepatitis B virus (Dang et al., 2006), as well as to inhibit the catalytic activity of HCV NS5B Polymerase (Tramontano et al., 2011) and HIV-1 RT (Tramontano et al., 2011; Higuchi et al.. 1991; Esposito et al., 2012a, 2011; Kharlamova et al., 2009) and integrase (IN) functions in biochemical assays (Tintori et al., 2015; Esposito et al., 2015). In particular, a number of anthraquinone derivatives were reported to affect both HIV-1 RDDP and RNase H RT-associated functions in biochemical assays, but were not active on viral replication in cell-based assays (Tramontano et al., 2011; Higuchi et al., 1991; Esposito et al., 2012b, 2011; Kharlamova et al., 2009). Hence, we wanted to evaluate the effects of the chemical components originated from the Rheum Chinese plants to inhibit the HIV-1 RT-associated and IN activities and to interfere with the HIV-1 life cycle in order to identify new scaffold inhibiting multiple HIV-1 targets.

Materials and methods

Plant materials and reagents

From June 2007 to September 2007, R. palmatum L. and R. officinale Baill. were collected from Gansu and Sichuan province respectively in China by one of the authors, and were naturally dried at room temperature. The raw materials were authenticated by Prof. Anren Li (Institute of Medicinal Plant Development, Beijing, China). The voucher specimens were deposited in the Laboratory of Pharmaphylogeny, Institute of Medicinal Plant Development (Beijing, China). The reference compounds AloeEmodin, Rhein, Emodin, Chrysophanol, Physcion were purchased from the National Institute for Control of Pharmaceuticals and Biological Products (Beijing, China), and Sennoside A and Sennoside B from the Mansite Pharmaceutical Co. Ltd. (Chengdu, China). The positive controls (S)-6-chIoro-4-(cyclopropylethynyl) 4-(trifluoromethyl)-1H-benzo[d][1,3]oxazin-2(4H)-one (Efavirenz) was purchase from Sigma-Aldrich (Milano, Italy), the (Z)-ethyl 4-(1-(2-chlorobenzyl)-1H-pyrrol-3-yl)-2-hydroxy-4- oxobut-2-enoate (RDS1759) was kindly provided from Prof. Di Santo (University of Roma La Sapienza) and N-[(4-fluorophenyl)methyl)-1,6-dihydro 5-hydroxy-l-methyl-2-[1-methyl-l-[[(5-methyl-l,3,4-oxadiazol-2-yl)carbonyl]amino]ethyl]-6-oxo-4-pyrimidinecarboxamide mono potassium salt (Raltegravir) was purchase from ChemScene (Monmouth Junction, NJ). HPLC grade acetonitrile and methanol were purchased from Burdick & Jackson (Muskegon, MI, USA). Deionized water was prepared by Milli-Q system (Millipore, Bedford, MA, USA). Phosphoric acid and methanol were of analytical grade purchased from Beijing Beihai Fine Chemicals Co. Ltd. (Beijing, China).

Preparation of sample solutions

The dried roots of plant materials were grounded by an electric mill into powder (40 mesh). Individual samples (~200 mg) were accurately weighed, then mixed with 30 ml methanol/water (30 ml, 80:20, v/v) and sonicated for 60 min at room temperature. The extract solution was cooled down to room temperature, and appropriate amount of the solution was added to the original weight. The final solution was filtered through 0.22 [micro]m membrane and then injected 10 [micro]l for each HPLC analysis (Fig. S1).

Dried roots (~1 g) of R. palmatum L, and R. officinale Baill. were extracted twice, with 150 ml methanol/water (80:20, v/v) and sonicated for 60 min at room temperature for each extraction. The extraction was together condensed and freeze-dried, with about 113 mg power harvesting.

Protein expression and purification

HIV-1 RT gene subcloned into the p6HRT_prot plasmid was kindly provided by Stuart Le Grice (NCI, Frederick, Maryland, USA). Protein expression and purification was performed in M15 Escherichia coli cells as described (Esposito et al., 2012a). Full-length IN and LE DGF proteins were expressed in BL21 E. coli (DE3) (Tintori et al., 2015; Esposito et al.. 2015). HIV-1 K103N, Y108C and Y188L RT mutants were produced by site-directed mutagenesis using the Stratagene kit (Agilent Technologies, Milano, Italy), according to manufacturer's indication as described (Corona et al., 2014a).

RNase H polymerase-independent cleavage assay

The HIV-1 RT-associated RNase H activity was measured as previously described (Esposito et al., 2013).

RDDP assay

The HIV-1 RT-associated RDDP activity was measured using the Enz-Check Reverse Transcriptase Assay Kit (Life technologies, Carlsbad. California, USA), as previously described (Corona et al., 2014a). The Yonetani-Theorell analysis was performed as previously reported (Esposito et al., 2011).

Homogeneous time resolved fluorescence (HTRF) LEDCF dependent and independent assay

The IN LEDGF/p75 dependent assay allowed to measure the inhibition of the 3' processing and strand transfer IN reactions in the presence of recombinant LEDGF/p75 protein, as previously described (Tintori et al., 2015; Esposito et al., 2015).

Cell line cultures

The human T-lymphoblastoid Jurkat cell line, clone E6-1, and the human embryonic kidney 293T cell line were obtained from ATCC (Manassas, VA, USA) and maintained in RPMl 1640 or DMEM (GIBCO, Life Technologies, Monza, Italy), respectively, supplemented with 10% heat-inactivated fetal bovine serum at 37[degrees]C in 5% C[O.sub.2] humidified atmosphere.

Plasmids and viruses

HIV-1 stock was produced by transient transfection of Jurkat cells with the pSVC21 plasmid containing the infectious HXBc2 molecular clone of HIV-1 (Ratner et al., 1985) by the DEAE-dextran method as described previously (Cullen, 1987) and stored at -80[degrees]C until use. Viral titer was measured as 50% Tissue Culture Infective Doses ([TCID.sub.50])/ml on C8166 cells by the Reed and Muench end point dilution method (Reed and Muench, 1938).

Recombinant HIV-1 HXBc2 CAT virus was produced by cotransfection of 293T cells with the pSVC21 [Vpr.sup.+][Vpu.sup.+][Nef.sup.+][DELTA]env-CAT plasmid along with the pSVIIlenv plasmid (J. Sodroski, Harvard University, Boston), with the Ca3(P04)2 method. The pSVC2.1 [Vpr.sup.+][Vpu.sup.+][Nef.sup.+][DELTA]env CAT plasmid (Sartori et al., 2011) was generated from the pSVC2.1 plasmid (J. Sodroski, Harvard University, Boston) which contains the complete proviral genome of the HIV-1 HXBc2 ([vif.sup.+]-[vpu.sup.-]-v[pr.sup.-]-[nef.sup.-]) (Ratner et al., 1985), by inserting the Vpu, Vpr and Nef encoding sequences and deleting the BglII-BglII env region. The chloramphenicol acetyltransferase (CAT) reporter gene was inserted in the pSVC2.1 [Vpr.sup.+][ .sup.+][Nef.sup.+][DELTA]env vector with resulting inactivation of the rev gene. The pSVIIIenv plasmid expresses the HIV-1 laboratory-adapted T-cell-tropic strain HXBc2 envelope glycoprotein along with Rev. Recombinant HIV-1 virions expressing CAT were collected 48 h post-transfection and filtered (0.45-pm-pore-size filter). Viral titer was determined in terms of Reverse Transcriptase (RT) activity (Rho et al., 1981).

HIV-1 env complementation assay

Jurkat cells (1 x [10.sup.6]) were incubated with 30,000 [sup.3H cpm RT units of recombinant CAT reporter virus at 37[degrees]C in the absence or presence of different amount of compounds (5, 10 and 20 [micro]M). Cells were lysed 4 days after infection and CAT activity was determined. Compound concentration required to inhibit early phases of HIV-1 replication by 50% ([EC.sub.50]) was calculated by nonlinear regression analysis with Sigma Plot (Jandel Scientific). Only results within a linear range (HIV-1 LTR-driven reporter CAT gene expression, i.e. conversion of chloramphenicol to acetyl chloramphenicol above 50%) were elaborated.

Cytotoxicity assay

The cytotoxicity of the compounds on the Jurkat cell line was assessed using the MTT method (Mosmann, 1993).

Quantitative real time PCR analysis of HIV-1 DNA

Jurkat cells (1 x [10.sup.6]) were infected with 30,000 [sup.3]H cpm RT units of HIV-1 HXBc2 CAT virus in the absence or presence of drugs (Raltegravir was used at 0.5 [micro]M. Efavirenz at 0.1 [micro]M, Sennoside A at 20 [micro]M, Sennoside B at 20 [micro]M and RDS1760 at 25 [micro]M). Total intracellular DNA was purified from treated and untreated cells 16 h post-infection with DNeasy blood and tissue kit (Qiagen, Limburg Netherlands), according to the manufacturer's instructions, and treated with 20 U/ml of Dpnl for 2 h at 37[degrees]C to remove contaminating plasmid DNA (Munir et al., 2013).

The early (negative strand strong stop) and late (gag) reverse transcription products and the total HIV-1 DNA were measured using quantitative real-time PCR (qPCR), as previously described (Yu et al., 2008; Doitsh et al., 2010; Cheney and McKnight, 2010). Integrated HIV-1 DNA was detected by nested Alu-gag PCR, as reported (Yu et al., 2008; Liszewski et al., 2009), with some modifications. Pre-amplification reaction was performed using AmpliTaq Gold DNA Polymerase (Applied Biosystems, Monza, Italy) and consisted of one cycle of denaturation (10 min 95[degrees]C), 39 cycles of amplification (15 sec 94[degrees]C, 30 sec 50[degrees]C, 8 min 72[degrees]C) and one cycle at 72[degrees]C for 7 min. Quantitative PCR reactions were performed using TaqMan universal PCR master mix (Applied Biosystems, Monza, Italy). The PCR consisted of one cycle of denaturation (10 min at 95[degrees]C) followed by 50 cycles of amplification (15 s at 95[degrees]C, 1 min at 60[degrees]C) in an ABI Prism[R] 7000 Sequence Detection System (Applied Biosystems, Monza, Italy).

Time of addition

Jurkat cells were infected with HIV-1 HIB at a multiplicity of infection (M.O.I.) of 0.5 [TCID.sub.50]/cell. After 1 h of incubation at 37[degrees]C, the cultures were washed twice and [10.sup.5] cells were maintained in the absence or presence of drugs. Test and reference compounds were added at different time points after infection (0, 1, 2, 3, 4, 5, 6, 7, 8 h). Virus production in the supernatant of infected cells was determined by p24 assay at 31 h post-infection, according to manufacter's instructions (Innotest[R]HIV Antigen mAb, Fujirebio, Ghent, Belgium). Raltegravir was used at 1 [micro]M, Efavirenz at 1.5 [micro]M, Zidovudine at 0.4 [micro]M, and Sennoside A at 250 [micro]M.

Results and discussion

Effect of extracts from rheum Chinese species and of their chemical components of both HIV-1 RT-associated functions

With the aim of identifying new scaffolds able to inhibit both HIV-1 RT-associated functions to be developed as novel antiviral drugs (Distinto et al., 2012, 2013), we thought that TCM could be a valid source of compound biodiversity. Hence we tested extracts from R. palmatum L. and R. officinale Baill., firstly for their ability to inhibit the HIV-1 RNase H activity, for which no drugs are currently available (Corona et al., 2013). Results showed that extracts from both plants potently inhibited this enzyme function with [IC.sub.50] values of 0.9 and 0.25 [micro]g/ml, respectively (Table 1).

Subsequently, single constituents of the extracts were purified by HPLC and seven phenolic components were found to be their major bioactive constituents (Table 1). Specifically, these components included anthraquinone derivatives such as Emodin, Crysophanol, Aloe-Emodin, Physcion, Rhein and Sennoside (both Sennoside A and Sennoside B). Hence, we investigated their ability to inhibit both HIV-1 RT-associated RNase H and RDDP functions using the RNase H selective diketo acid (DKA) derivative DS1759 (Corona et al., 2014a) and the NNRTI Efavirenz as controls (Table 1). Physcion and Emodin were not significantly active on both HIV-1 RT-associated RDDP and RNase H activities (as the maximum concentration tested they inhibited slightly less than 50& of enzyme activity), while Aloe-Emodine and Crysophanol inhibited both HIV-1 RT-associated activities with [IC.sub.50] values around 21-26 [micro]M (Table 2). Differently. Rhein inhibited weakly the HIV-1 RT-associated RNase H function, but not its RDDP activity. Noteworthy, the most active components were Sennoside A and Sennoside B. that inhibited both HIV-1 RT-associated functions with [IC.sub.50] values in the 2-5 [micro]M range.

Evaluation of the effects of Sennoside A and B on mutated RT

Since Sennosides A and B were identified as novel dual functions RTIs, we wanted to compare them to known NNRTIs or RNase H inhibitors by using a series of previously described HIV-1 RT mutants. Thus, we firstly tested the effect of Sennoside A and B on three known single RT mutants involved in NNRTI resistance such as K103N, Y181C and Y188L RTs, using Efavirenz as positive control (Table 3). Results showed a significant difference between Sennoside A and B. In fact, in the presence of these mutations Sennoside A showed a 4- to 15-fold decrease in its potency of inhibition. On the contrary, Sennoside B exhibited a 2- to 4-fold decrease in the inhibitory activity on K103N and Y188L RTs, and displayed an [IC.sub.50] value on Y181C RT similar to the one obtained on wild type RT (Table 3). Next, we tested the effects of Sennoside A and B on the RNase H activity of RTs bearing mutation in the 474 and 475 amino acid residues (N474A and Q475A RTs) that are highly conserved and were recently reported to be involved in the selective binding of the DKA derivative RDS1759 (Kharlamova et al., 2009; Corona et al., 2014a, 2014b). Results indicated that, compared to wild type RT, the inhibitory activity of both compounds were affected by the presence of the two mutations, leading to a decrease in their efficacy by 5- to 12-fold (Table 3). Thus, in the case of RNase H mutants, Sennoside A and B displayed a similar reduction in the inhibitory activity.

Subsequently, considering the results obtained with Sennoside A on the NNRTI resistant RT mutants, we wanted to better characterize its behavior in the presence of Efavirenz in order to explore the possibility that the two compounds may interact with the same RT binding site. Therefore we tested the effect of increasing concentrations of Sennoside A and Efavirenz on the RT-associated RDDP activity and analyzed the data by a Yonetani-Theorell plot (Yonetani, 1982) (Fig. 1). Such analysis can reveal whether simultaneous binding (or inhibition) of the HIV-1 RT enzyme by the two compounds is occurring or not. Results showed that Sennoside A and Efavirenz are kinetically mutually exclusive, suggesting that either they bind to the same site or the binding of one compound prevents the binding of the second one.

Taken together, this first set of data indicated that Sennoside A and B inhibit both RT-associated functions. In particular, Sennoside A showed a reduced ability to affect the RDDP activity of K103N, Y181C and Y188L RT mutants, similarly to Efavirenz, and kinetic analysis indicated that Sennoside A and Efavirenz are kinetically mutually exclusive. Overall, these results suggest that Sennoside A binding might involve the NNRTIs binding pocket, and thus inhibit the RT-associated RDDP function binding to this pocket. However, it is well known that Efavirenz and other NNRTIs increase RT-associated RNase H activity (Palanappian et al., 1995; Radzio and Sluis-Cremer, 2008), while our biochemical studies showed that Sennoside A inhibits this function. The observation that Sennoside A is less effective in inhibiting the RNase H activity in the presence of N474A and Q475A mutations, that were previously reported to strongly affect DKA derivatives binding and inhibitory activity (Corona et al., 2014a), suggests that Sennoside A could also recognize a second site that might be localized nearby or within RNase H domain. Overall, the binding of Sennoside A to the first site, in the polymerase domain, would lead to the inhibition of the HIV-1 RT-associated RDDP function, while the binding to the second site, in the RNase H domain, would lead to the inhibition of the HIV-1 RNase H function.

Evaluation of the effects of Sennoside A and B on HIV-1 IN activity

Since a number of DKA derivatives inhibiting the RNase H activity by binding to the RNase H domain were reported to inhibit the HIV-1 IN activity (Esposito et al., 2012; Esposito et al., 2011; Tintori et al., 2015; Esposito et al., 2015; Tramontano et al., 2005; Costi et al., 2013; Costi et al., 2014; Cuzzuculi Crucitti et al., 2014), we asked whether Sennoside A and B might affect also the IN catalytic function. Thus, we tested their ability to inhibit the HIV-1 IN strand transfer reaction in the presence of the LEDGF/p75 cellular cofactor, using Raltegravir as positive control (Table 3). Data showed that Sennoside A inhibited the HIV-1 IN strand transfer activity with an [IC.sub.50] value of 3.8 [micro]M, while Sennoside B was 23-fold less potent.

Pharmacophoric differences between Sennoside A and B

Sennoside A and B were shown to differently affect HIV-1 wild type and mutant RTs as well as IN strand transfer catalytic function. Hence, in order to investigate the different behavior of the two isomers A (Threo) and B (Erythro), we calculated their global minimum energy conformation (Table S1). Sennoside A and B are optical stereoisomers and therefore they do not differ for their chemical properties; however their pharmacophoric features are diverse for their spatial orientation (Fig. 2A and B). These overall sterical differences can explain the different performance of the two Sennoside stereoisomers. In this respect, considering the large molecular surface of both Sennoside molecules, the different localization of wide hydrophobic areas in the two stereoisomers can dramatically affect the potential establishment of hydrophobic bonds and therefore influence the biological activity.

Characterization of the mechanism of action of Sennoside A and B in cell-based assays

Given their ability to inhibit both the HIV-1 RT and IN functions in biochemical assays, we wanted to evaluate the effect of Sennoside A and B on the early phases of the HIV-1 replication. Thus, we selected a transient trans-complementation assay to assess the replicative potential of HIV-1 in a single round of infection using a lymphoblastoid T cell line as target (Helseth et al., 1990; Parolin et al., 2003). In this assay, an env-defective HIV-1 HXBc2 [Vpr.sup.+][Vpu.sup.+][Nef.sup.+] [DELTA]env provirus encoding the bacterial CAT gene was complemented by the envelope glycoprotein from the HXBc2 laboratory-adapted T-tropic virus. The level of CAT expression in the cell lysates allowed us to examine early events in the infection process (Fig. 3). Results showed that Sennoside A significantly inhibited HIV-1 replication with an [EC.sub.50] value around 9 [micro]M in the absence of cytotoxicity, as determined by MTT assay, while Sennoside B was inactive at the highest tested concentration. In addition Crysophanol, even though it was able of inhibiting both HIV-1 RT-associated activities in biochemical assay (Table 2), did not exert any effect on HIV-1 replication.

In order to identify the viral process targeted by Sennoside A, we examined its effect on both reverse transcription and integration process by quantitative real time PCR (qPCR) analyzing viral cDNA intermediates in Jurkat infected cells treated with the drug. The primers and probe set used were selected in order to monitor sequential steps within the reverse transcription process, corresponding to generation of negative strong-stop DNA (early) and DNA strand elongation (late), and to quantify viral DNA that have successfully completed the integration process (Fig. 4A). Moreover, we assessed total viral DNA synthesis, including reverse transcription products that have completed the second strand transfer, as well as linear and circular form of viral DNA and integrated DNA (Fig. 4A). Compounds were added at the time of infection and DNA collected at 16 h post-infection. Efavirenz, Raltegravir and the DKA derivative RDS1760 (Corona et al., 2014a) were used as positive controls (Fig. 4B). Results showed that negative strong stop DNA synthesis was only slightly inhibited by Sennoside A at 20 [micro]M concentration (Fig. 4B). In contrast, late reverse transcription products generated after the first strand transfer decrease in the presence of Sennoside A, but not in the presence of Sennoside B. The reduction of total viral DNA synthesis further confirmed this result.

Based on these observations, Sennoside A seemed to exhibit a mechanism of action similar to the one displayed by Efavirenz, affecting the RT activity during or soon after the first template exchange. Integration assay with an Alu-gag qPCR revealed that treatment with Sennoside A reduced also viral integration. Next, in order to gain further insights into the mechanism of action of Sennoside A, we performed a time of addition study in which compounds were added at the time of infection and every hour up to 8 h after infection, using Efavirenz, Zidovudine and Raltegravir as controls (Fig. 5). Jurkat cells were infected with HIV-1 (Pauwels et al., 2011) and compounds were added with time lags of up to 8 h after infection. According to the target of drug action or to the stage of interaction, addition of compound could be delayed for a specific number of hours without loss of antiviral activity. Results showed that Sennoside A profile of inhibition was similar to the one observed for the nucleoside analog Zidovudine (Fig. 5). These data confirm RT as a target of interaction of Sennoside A. On the other hand, our experimental setting cannot exclude that Sennoside A might have a slight effect also on HIV-1 IN.

Conclusions

Starting from the TCM heredity and knowledge, we successfully identified in the Rhubarb the chemical component Sennoside A that is able to inhibit in the low micromolar range both HIV-1 RT-associated activities, IN function and virus replication. Mode of action studies performed in cell cultures, demonstrated that Sennoside A targets the HIV-1 reverse transcription process, while it seems to be unable to significantly affect IN activity. Overall, Sennoside A represents a novel attractive scaffold of dual function RTI that deserves further investigations by means of chemical modification, in search of new dual enzyme derivatives active on both HIV-1 RT and IN. These results also suggest that Rhubarb might be a good, natural addition to the diet of HIV positive patients.

ARTICLE INFO

Article history:

Received 26 April 2016

Revised 19 July 2016

Accepted 9 August 2016

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments

This work was supported by RAS grant LR 7/2007 CRP-24915, by Istituto Superiore di Sanita grant n. 40H98, and by 60% grant from the University of Padova.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.08.001.

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Francesca Esposito (a), liaria Carli (b,1), Claudia Del Vecchio (b,1), Lijia Xu (c), Angela Corona (a), Nicole Grandi (a), Dario Piano (a), Elias Maccioni (a), Simona Distinto (a), Cristina Parolin (b,2), **, Enzo Tramontano (a,d), *

(a) Department of Life and Environmental Sciences. University of Cagliari. Cittadella di Monserrato SS554, 09042. Monserrato. Cagliari, Italy

(b) Department of Molecular Medicine. University of Padova. via Gabeiii 63. 35121 Padova. Italy

(c) Institute of Medicinal Plant Development (IMPLAD). 151 Malianwa North Road Haidian District. 100193 Beijing, China

(d) Genetics and Biomedical Research institute. National Research Council (CNR). Cittadella di Monserrato SS554. 09042, Monserrato. Cagliari. Italy

Abbreviations: DKA, diketo acid; [EC.sub.50]. HIV-1 replication by 50%: HAART. Highly Active Anti-Retroviral Therapy; HIV-1. Human Immunodeficiency Virus type 1; IN. integrase; INIs, HIV-1 IN inhibitors; NNRTI, Non-Nucleoside RT Inhibitor; NRTIs, Nucleoside RT Inhibitors; PI. Protease Inhibitor; qPCR, quantitative real-time PCR; RDDP, Reverse Transcriptase (RT)-associated DNA Polymerase: RNase H, Ribonuclease H; [TCID.sub.50] 50%/ml. Tissue Culture Infective Doses; TCM. Traditional Chinese Medicine.

* Corresponding author. Fax: +39 0706754536.

** Corresponding author.

E-mail addresses: cristina.parolin@unipd.it (C. Parolin), tramon@unica.it (E. Tramontano).

(1) Authors contributed equally to the paper.

(2) Fax: +39 049 8272355.

http://dx.doi.Org/10.1016/j.phymed.2016.08.001

Table 1
Description of Species, collecting place, origin and
components of Rheum Chinese and their effects on HIV-1
RNase H.

Species              Place of           Origin   Aloe Emodin   Rhein
                     collection                  (mg/g)        (mg/g)

R. palmamm (D-13)    Tianzhu, Cansu     Wild     0.023         --
R. officinale        Songpan. Sichuan   Wild     0.05          0.039
  (D-15)

Species              Emodin   Chrysophanol   Physcion   Sennoside A
                     (mg/g)   (mg/g)         (mg/g)     (mg/g)

R. palmamm (D-13)    0.046    0.055          0.102      0.864
R. officinale        0.033    0.107          0.16       0.162
  (D-15)

Species              Sennoside B   HIV-1 RNase H
                     (mg/g)        (a) [IC.sub.50]
                                   ([micro]g/ml)

R. palmamm (D-13)    0.489         0.9
R. officinale        0.037         0.25
  (D-15)

(a) Extract concentration required to inhibit the
HIV-1 RT-associated RNase H activity by 50%.

Table 2
Effect of Rheum single components on the
HIV-1 RT-associated activities.

Compound        Chemical       (a) I[C.sub.50] ([micro]M)
                structure
                               RNase H             RDDP

Emodin          [FORMULA NOT   >100(59%) (b)       >100(51%)
                REPRODUCIBLE
                IN ASCII.]

Chtysophanol    [FORMULA NOT   25.0 [+ or -] 4.0   26.5 [+ or -] 2.5
                REPRODUCIBLE
                IN ASCII.]

Aloe-Emodin     [FORMULA NOT   23.0 [+ or -] 5.0   21.0 [+ or -] 4.0
                REPRODUCIBLE
                IN ASCII.]

Physcion        [FORMULA NOT   > 100 (53%)         > 100 (57%)
                REPRODUCIBLE
                IN ASCII.]

Rhein           [FORMULA NOT   60 [+ or -] 12      >100 (62%)
                REPRODUCIBLE
                IN ASCII.]

Sennoside A     [FORMULA NOT   1.9 [+ or -] 0.3    5.3 [+ or -] 0.1
                REPRODUCIBLE
                IN ASCII.]

Sennoside B     [FORMULA NOT   2.1 [+ or -] 0.2    2.3 [+ or -] 0.4
                REPRODUCIBLE
                IN ASCII.]

Efavirenz       [FORMULA NOT   >50 (c)             0.025 [+ or -]
                REPRODUCIBLE
                IN ASCII.]
                                                     0.0005 (c)
RDS1759                        7.4 [+ or -]        > 50 (c)
                                 0.2 (c)

(a) Compound concentration required to reduce HIV-1
RT-associated RNase H and RT-associated RNA Dependant
DNA Polymerase activities by 50%.

(b) Percentage of control activity in the presence of 100
[micro]M concentration of compound.

(c) Data were expressed in [micro]M.

Table 3
Effect of Sennoside A and B on mutated HIV-1 RT-associated
and HIV-1 IN activities.

Compounds      RDDP I[C.sub.50]
               ([micro]M) (a)

               K103N RT                    Y181C RT

Sennoside A    78 [+ or -] 1 (14.7) (d)    213 [+ or -] 2.1 (4.0)
Sennoside B    8.7 [+ or -] 0.2 (3.7)      2.5 [+ or -] 03 (1.1)
Efavirenz      0.19 [+ or -]  0.02 (7.6)   0.06 [+ or -] 0.02 (2.4)
RDS1759        >50                         >50
Raltegravir    >50                         >50

Compounds      RDDP I[C.sub.50]           RNase H I[C.sub.50]
               ([micro]M) (a)             ([micro]M) (b)

               Y188L RT                   N474A RT

Sennoside A    64 [+ or -] 2 (12.1)       18.4 [+ or -] 3.7 (9.2)
Sennoside B    6.6 [+ or -]  1.2 (2.9)    10.8 [+ or -]  1.7 (5.1)
Efavirenz      0.21 [+ or -] 0.03 (8.4)   >50
RDS1759        >50                        90 [+ or -] 5 (12.2)
Raltegravir    >50                        >50

Compounds      RNase H I[C.sub.50]        IN I[C.sub.50]
               ([micro]M) (b)             ([micro]M) (c)

               Q475A RT                   wt IN

Sennoside A    17.7 [+ or -] 1.5 (8.8)    3.8 [+ or -] 0.4
Sennoside B    24.8 [+ or -] 0.8 (11.8)   87 [+ or -] 12
Efavirenz      >50                        >50
RDS1759        94 [+ or -] 6 (12.3)       >50
Raltegravir    >50                        0.066 [+ or -] 0.010

(a) Compound concentration required to reduce HIV-1
RT-associated RNA Dependant DNA Polymerase activity by 50%.

(b) Compound concentration required to reduce HIV-1
RT-associated RNase H activity by 50%.

(c) Compound concentration required to inhibit the HIV-1 IN
catalytic activities by 50% in the presence of LEDCF.

(d) Number in parenthesis indicate the fold of I[C.sub.50]
value increase with respect to HIV-1 wt RT (results shown in
Table 1).


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Title Annotation:Original article
Author:Esposito, Francesca; Carli, Ilaria; Del Vecchio, Claudia; Xu, Lijia; Corona, Angela; Grandi, Nicole;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Nov 15, 2016
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